Exercise-induced dehydration alters pulmonary function but does not modify airway responsiveness to dry air in athletes with mild asthma

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INTRODUCTION
Whole-body dehydration commonly occurs in athletes engaging in endurance events (36), with loss of body mass averaging 2.3% after marathons (41) and 2.9% after ultramarathons (35).Whole-body dehydration is thought to limit exercise performance due to strain on multiple organ systems, including the circulatory, (central) nervous, muscular, integumentary, and urinary systems (8,37).Lung fluid balance and water transport at pulmonary surfaces play an important physiological role in the maintenance of airway hydration and in proper airway clearance (19).Relatively little is known however about the impact of whole-body dehydration on the respiratory system.
Early studies (15,18) produced conflicting results on the effects of whole-body dehydration on pulmonary function in the healthy human.A reduction in forced expiratory volume in one second (FEV 1 ) was initially observed in mildly dehydrated individuals following fluid deprivation (15), suggesting that whole-body dehydration is associated with airflow limitation.However, after diuretic drug administration resulting in moderate dehydration, an improvement in expiratory flow rates, including FEV 1 , was subsequently noted (18).Therefore, uncertainty remains as to the impact of whole-body dehydration on the healthy human lung.
Equally uncertain is whether whole-body dehydration constitutes a significant risk factor for broncho-pulmonary disorders (19).A large body of evidence points toward acute dehydration of the airway surface liquid as a key determinant of exercise-induced bronchoconstriction (EIB) (2).EIB is characterized by a transient narrowing of the airways (with associated reduction in expiratory airflow) in response to vigorous exercise.
Presence of EIB is established through serial measurements of FEV 1 after exercise (or dry air hyperpnea), with a fall in FEV 1 ≥10% used as the diagnostic criterion (31).EIB is thought to occur in response to osmotic and thermal stress within the airways during exercise-hyperpnea (2).Water and heat are lost from the airway surface in response to humidifying large volumes of inspired (unconditioned) air over a short period of time (12).
The evaporative water loss is proposed to increase the osmolarity of the airway surface liquid -particularly at the level of the small airways (11) -.This would then stimulate the release of broncho-active mediators and cause, in susceptible individuals, the airway smooth muscle to contract (2).
Endurance athletes and patients with asthma are at increased risk for EIB (13,20).Airway surface dehydration may be exacerbated during exercise in both populations: in athletes, as a result of the high ventilatory demand associated with strenuous exercise; and in patients with asthma, due to dysregulation of body fluid secretions (30).Bronchial circulation is the primary provider of fluid to the airways.Since exercise-induced dehydration causes hypovolemia and increases blood plasma osmolarity (8), alterations in the volume and composition of bronchial blood flow are to be expected in a state of dehydration.Whole-body dehydration may therefore diminish airway surface hydration, resulting in an amplified bronchoconstrictive response to exercise.
The primary aim of this study was to establish the impact of exercise-induced dehydration on airway responsiveness in athletes with a prior medical diagnosis of mild asthma and/or EIB.Our hypothesis was that the fall in FEV 1 after dry air hyperpnea would be exacerbated in a state of mild dehydration.Since the effect of whole-body dehydration on resting pulmonary function remains uncertain, we also assessed pulmonary function, via spirometry, whole-body plethysmography and diffusing capacity for carbon monoxide, before and after induced dehydration.

Participants
Ten recreational athletes, aged 18 to 35 y, participated in the study.All participants had a prior doctor diagnosis of mild asthma and/or EIB and reported respiratory symptoms (chest tightness, wheeze, mucus hyper-secretion, cough) during and/or after exercise.
Participants taking any asthma medication other than inhaled short acting β 2 -agonists or anti-histamines were excluded.Those on medication(s) were required to withhold inhaled short-acting β 2 -agonists for a minimum of 8 h and anti-histamines for 72 h prior to the visits (4).Participants were non-smokers, free from respiratory infections for 4 wk prior to the study, and had no known chronic medical condition other than asthma or EIB.All participants provided written informed consent.The institutional research ethics committee approved the study (ref# .

Protocol
The study used a randomized crossover design with three experimental visits.Pulmonary function was assessed using spirometry, whole-body plethysmography and diffusing capacity before and up to 2 h after each of the following conditions: i) exercise with no fluid intake (dehydration); ii) exercise with ad libitum fluid intake (control); iii) time-matched rest period (rest).The 'rest' condition controlled for the possible confounding effect of airway refractoriness after the exercise bout that was used to dehydrate the participants (24).
Airway responsiveness was assessed 2 h after the completion of exercise/rest using a standard eucapnic voluntary hyperpnea (EVH) challenge with dry air (1).To determine whether any changes caused by dehydration could be quickly reversed, a rehydration phase was included in the dehydration condition of the study.In that condition, participants were allowed to drink water ad libitum between 20 and 60 min after the EVH challenge, after which final spirometry testing was performed.A schematic of the experimental protocol is presented in Figure 1.
All visits commenced in the morning so as to standardize for diurnal variation in pulmonary function (38).Participants were asked to withhold alcohol, caffeine and exercise on the day of testing.

Hydration status
Participants were asked to arrive at each experimental visit in a euhydrated state.Upon arrival, urine osmolality was measured using a portable refractive index osmometer (Osmocheck, Vitech Scientific Ltd, UK).Adequate hydration was defined as <700 mOsmol•kgH 2 O -1 (36).Nude body mass was recorded before and 60 min after exercise or time-matched rest using a calibrated scale (SECA model 798, Hamburg, Germany), with the change in body mass used as an index of dehydration.

Exercise period
In the control and dehydration conditions, participants completed 2 h of low intensity exercise.The exercise involved four bouts of 20 min of cycling, with each bout followed by 10 min of stepping.Cycling was performed at 25% of estimated peak power (16).
Stepping was conducted on a 20 cm step at a rate of 45 steps per minute.Mid-way through each bout of exercise, heart rate was measured using telemetry (Polar H7, Polar Electro (UK) Ltd, Warwick, UK) and minute ventilation using offline gas analysis (Douglas bags and Harvard dry gas meter).To induce dehydration, exercise was performed in an environmental chamber (Procema Ltd, Twickenham, UK) set at 37 °C and 50% relative humidity and fluid intake was prohibited.In the control condition, environmental temperature was set at 20 °C (ambient humidity) and fluid consumption was ad libitum.In the rest condition, participants remained seated in ambient conditions and were allowed to consume fluid ad libitum.

Pulmonary function tests
Pulmonary function was assessed using a commercially available system (Masterscreen, CareFusion, Hochberg, Germany).Spirometry was conducted at baseline and at 10 min and 120 min after exercise (or rest).Forced expiratory maneuvers were performed in accordance with ATS/ERS guidelines (28).Measurements were performed in triplicate, and the largest FEV 1 and FVC from reproducible maneuvers (i.e., between-maneuver differences <150 ml for FEV 1 and FVC) were kept for analysis.Following the EVH challenge, expiratory maneuvers were performed in duplicate (1).The GLI-2012 equations (34) were used for calculation of predicted values and lower limits of normal.
Whole-body plethysmography was used to determine static lung volumes and capacities according to ATS/ERS guidelines (39).Measurements were performed at baseline and at 60 min after exercise (or rest).The mean of three reproducible trials (i.e., the three functional residual capacity [FRC] values agreeing within 5%) was used for analysis.
Residual volume (RV) was derived from the mean FRC minus mean expiratory reserve volume (ERV), and TLC was calculated as the sum of maximum vital capacity (VC) and RV.
Diffusing capacity of the lung for carbon monoxide (DLCO) was assessed using the singlebreath technique according to ATS/ERS guidelines (26).The measurements were performed at baseline and at 90 min after exercise (or rest).The maneuver was repeated at least twice to ensure repeatability (i.e., <10% variation in DLCO).The mean DLCO, transfer coefficient (KCO), and alveolar volume (VA) were calculated from two reproducible maneuvers and used for analysis.Diffusing capacity data for one participant was lost due to technical error.

Airway responsiveness
Airway responsiveness to dry air was assessed via EVH (1).Briefly, participants were asked to breathe for 6 min at a target ventilation of 85% predicted maximum voluntary ventilation (MVV, estimated as 30 × baseline FEV 1 ).A dry gas mixture of 21% O 2 , 5% CO 2 , and N 2 balance was delivered by a commercially available system (Eucapsys, SMTEC, Nyon, Switzerland).Ventilation was measured throughout the test, with participants receiving real-time visual feedback.The ventilation achieved during the first visit was set as the target ventilation for subsequent visits.Before and at regular time points after EVH (2, 5, 10, 15, 20 and 60 min), forced expiratory maneuvers were performed, with the maximum percentage change in FEV 1 from 'baseline' (i.e., the value recorded immediately pre-EVH) used as the index for airway responsiveness.A sustained ≥10% fall in FEV 1 (over two consecutive time-points) was consistent with a diagnosis of EIB (4).

Statistics
Sample size was based on previous studies that have investigated the effect of dehydration on pulmonary function (18) and on those that have tested the effect of EVH on airway caliber in recreationally active individuals (7,21).All data were analyzed using statistical software (SPSS 20, Chicago, IL, US).Statistical significance was set at P<0.05 (unless otherwise stated).Data were tested for normality using the Shapiro-Wilk test.Data for the maximum fall in FEV 1 post-EVH were not normally distributed; therefore, differences between conditions were tested using a Friedman 2-way ANOVA by ranks test and data displayed as median and interquartile range (Q1-Q3).Resting spirometry, wholebody plethysmography and diffusing capacity data were normally distributed.Differences in resting pulmonary function between conditions and across times were analyzed using repeated-measures ANOVA with Bonferroni post-hoc analysis, as needed, and data are presented as mean ± SD.Heart rate and ventilation were averaged over the entire period of exercise and compared between dehydration and control conditions using paired t-test.
Relationships between changes in body mass (kg) and pulmonary function (l) in the dehydration condition were assessed using Pearson's correlation coefficient.

Participant characteristics
Ten recreational athletes (five female) completed the study.Mean age, height and body mass were: 21 ± 2 yr, 170 ± 12 cm and 63 ± 10 kg, respectively.Participants were involved in summer sports and trained for 6 ± 4 h per week in aerobic activities.Five participants had childhood asthma, and eight were using short acting β 2 -agonist medication at the time of the study.All participants had baseline FEV 1 and FVC above the lower limit of normal (34).

Hydration status
Baseline body mass was not different across conditions (P=0.74).The dehydration intervention caused a significant reduction in body mass (63.3 ± 10.4 kg at baseline vs. 61.8 ± 10.1 kg post-exercise, P<0.001), which equated to a loss of 2.3 ± 0.8%.There was no change in body mass following exercise in the control condition (63.3 ± 10.5 kg at baseline vs. 63.1 ± 10.5 kg post-exercise, P=0.085) or over the rest period (63.2 ± 10.8 kg at baseline vs. 63.0 ± 10.7 kg post-rest, P=0.12).Over the rehydration period in the dehydration condition, participants drank 830 ± 190 ml of water (61 ± 19% of the loss in body mass).

Exercise period
As expected, heart rate was significantly higher during exercise in the dehydration condition compared to the control condition (148 ± 16 vs.118 ± 20 bpm, respectively; P<0.001).Ventilation did not differ significantly between conditions (42 ± 15 l⋅min -1 in the dehydration condition vs. 34 ± 6 l⋅min -1 in the control condition; P=0.084).

Airway responsiveness
Participants achieved a mean ventilation of 104 ± 29 l⋅min -1 during the EVH challenge over the three experimental visits, which corresponded to 70 ± 9% of predicted MVV.No difference in ventilation was noted across conditions (P=0.64).Seven participants (70%) had an EVH response consistent with a diagnosis of EIB in at least one condition.One additional participant had a transient fall in FEV 1 during one visit.The median and interquartile range for maximum reduction in FEV 1 post-EVH was: 13% (7 -15%), 11% (9 -24%) and 12% (7 -20%) in the dehydration, control and rest condition, respectively (Figure 2).These values were not different between conditions (P=0.20).

Dynamic lung indices
At the start of the experimental visits, pulmonary function was not different between conditions (Table 1).However, a significant interaction effect over time was noted between conditions (P<0.001), with significant reductions in FVC only in the dehydration and control conditions (P<0.001 and P=0.014, respectively).In the dehydration condition, there was a sustained fall in FVC from baseline between 10 and 120 min of recovery [5.09 ± 1.22 l at baseline to 4.79 ± 1.10 l at 10 min (P=0.001)and 4.89 ± 1.10 l at 120 min post-exercise; (P=0.024)], while in the control condition the reduction in FVC was only transient (i.e., noted only at 10 min post-exercise) (Table 1).Further, the magnitude of change in FVC was greater under dehydration compared to both the control and rest conditions (Figure 3).
In a state of dehydration, eight participants (80%) presented a clinically meaningful reduction in FVC (>200 ml), whereas only one participant demonstrated a >200 ml fall in FVC in the control condition and none in the resting condition.Following rehydration, FVC remained slightly, but significantly, lower than baseline (−90 ± 100 ml, P=0.022).No significant differences were noted between times and/or conditions for FEV 1 and PEF (Table 1).

Static lung volumes and capacities
Static lung volumes and capacities at baseline were not different between-conditions (Table 2).Significant interaction effects were noted over the experimental conditions for FRC (P=0.004) and RV (P=0.001).In the dehydration condition, a significant increase in FRC was noted pre-to post-exercise (260 ± 250 ml, P=0.011); no difference was observed in the control or resting conditions (Table 2).A concurrent increase in RV of 260 ± 182 ml was observed under the dehydration condition (P=0.001)(Table 2).The magnitude of change in FRC and RV from pre-to post-exercise was greater under dehydration compared to control (P=0.015 and P=0.060, respectively).Further, the change in RV was greater under dehydration compared to rest (P=0.005)(Figure 4).No significant changes were noted between times and/or conditions for ERV or TLC (Table 3).Consequently, RV/TLC was increased under dehydration (P<0.001)(Table 3).

Diffusing capacity
There were no differences in baseline DLCO, KCO or VA between conditions.Further, our conditions did not modify any of these variables (Table 3).

Correlation analysis
There was a significant negative correlation (r=−0.703,P=0.023) between the change in body mass and the change in RV at 60 min post-exercise in the dehydration condition (Figure 5).No other significant relationships were noted between study variables.

DISCUSSION
The aim of this study was to investigate the effects of exercise-induced dehydration on airway responsiveness and pulmonary function in athletes with a medical diagnosis of mild asthma and/or EIB.We showed that mild dehydration does not increase airway responsiveness to dry air hyperpnea, but is associated with alterations in lung volumes (i.e., reduced FVC and increased FRC and RV).Mild whole-body dehydration is therefore unlikely to put athletes at increased risk for EIB.However, perturbations at the level of the small airways are likely to occur when athletes with pre-existing lung condition become dehydrated.
This study is the first to assess the effect of whole-body dehydration on airway responsiveness.Given that athletes regularly experience exercise-induced dehydration (41) and that EIB is the most common chronic condition in elite sport (13), these findings are highly relevant.We reasoned that whole-body dehydration may have the potential to affect the volume and/or composition of airway surface liquid and, consequently, could enhance the osmotic stimulus responsible for EIB (2).However, considering that no difference in the severity of bronchoconstriction was noted following EVH between our dehydration and control conditions, our data do not support our hypothesis.
To maintain ecological validity, we aimed to induce a state of mild dehydration using exercise.We were successful in that the average body mass loss was of 2.3%.A loss of 2% in body mass is believed to be the threshold for impairment of endurance performance via, amongst others, contraction of blood volume, increase in heart rate and high relative effort for a given workload (8).However, the mild degree of dehydration noted in the current study may have been insufficient to interfere with the pathophysiology of EIB.The volume of airway surface liquid is very small, with <0.5 ml of liquid covering the first seven generations of airways (5).Relative to the small volume of water available at the airway surface, water loss occurring within the airways during hyperpnea of dry air is very high.
Based on mathematical modeling, the net water loss within the airways during ventilation at 60 l•min -1 in temperate conditions (i.e., 26.7 °C and 8.8 mg•H 2 O•L -1 ) can exceed 0.4 ml•min -1 (12).In the present study, ventilation during EVH averaged 104 l•min -1 and inspired air (delivered through medical air canisters) was completely dry, consequently the net loss would be close to 1 ml•min -1 .It is therefore possible that the large volume of respiratory water loss during EVH negated any changes in airway surface liquid induced by our dehydration protocol.An alternative explanation for airway responsiveness being unaffected by whole-body dehydration is that the EVH test provoked a maximal airway response.A maximum response plateau is commonly observed during methacholine and histamine challenges in healthy individuals and patients with mild asthma (17,40).Similarly, a maximum response plateau has been shown to occur following bronchial provocation with exercise in children with asthma, with no further increase in the severity of EIB past six minutes of exercise (14).This raises the possibility that the use of EVH as bronchial stimulus may have masked the effects of whole-body dehydration on airway responsiveness.To address this issue, future work should be conducted using a dose-response bronchial challenge, such as the mannitol test (25).Further, since a maximal response plateau occurs less frequently in individuals with a greater degree of airway responsiveness (40), our findings should not be generalized to individuals with moderate-to-severe asthma/EIB.
Our study used two control conditions, whereby hydration was maintained during exercise and a rest period.The purpose of the rest condition was to rule out airway refractoriness as a confounding variable.Given that i) ventilation during exercise remained relatively low, ii) that FEV 1 and PEF were not different to baseline at 10 min post-exercise in either the control or dehydration condition, and iii) that no difference in airway responsiveness was noted between the two control conditions, we can be confident that the exercise protocol used to induce dehydration did not cause airway refractoriness and therefore interfere with our results.
A concurrent aim of our study was to establish the effect of exercise-induced dehydration on basal pulmonary function.In contrast to previous research (15,18), our results suggest that dehydration causes a reduction in FVC (with no associated change in FEV 1 ).
Previously, induced dehydration, by either fluid deprivation (15) or diuretic drug administration (18), had no effect on FVC.However, both types of intervention caused either a decrease (15) or an increase (18) in FEV 1 .The divergence in pulmonary function results in response to dehydration may be due to the various protocols.Fluid deprivation for 16 h resulted in smaller decreases in body mass than noted in the current study (range: 0.0 to 2.5% (15) vs. 1.5 to 4.4% in our study).While a more pronounced state of dehydration was induced by diuretics (∼4.5 % loss of body mass) (18), the different types of water loss (hypertonic vs. isosmotic) may have contributed to the discrepancy in the findings.Exercise-induced dehydration is well known to increase plasma osmolarity, whereas dehydration induced via diuretic administration generally results in isosmotic hypovolemia (37).In the present study, the increase in plasma osmolarity might have caused a redistribution of fluid away from the airways, which, in turn, may have affected lung volumes.That an inverse relationship was found in a large (>10,000) adult population between serum osmolarity and FVC (33) supports the idea that hypertonic dehydration could adversely affect pulmonary function.
In our study, the reduction in FVC was associated with a concomitant increase in RV, FRC and RV/TLC ratio; the latter a marker of air trapping (22).Further, a positive association was found between the degree of dehydration (as inferred by the reduction in body mass) and the increase in RV.Together, these results suggest that exercise-induced dehydration primarily affects the small airways.We propose that the main underlying mechanism for these changes is reduced peripheral airway stability caused by a change in the properties and/or volume of airway surface liquid in a dehydrated state.Airway surface liquid has low surface tension, which inhibits small airway closure at low lung volumes (27).If exerciseinduced dehydration increases airway surface tension, it would explain the reduction in FVC and the increase in RV.At present, there are no data pertaining to the effect of airway surface dehydration on surface tension; however, cystic fibrosis (a condition that causes depletion of airway surface liquid) may offer a reasonable model.Inhalation of dry powder mannitol -a treatment known to increase the secretion of fluid to the airway surface (9)has been shown to reduce surface tension of mucus secretions (10).It is reasonable to speculate, therefore, that the reduced FVC and increased RV under dehydration in the present study were caused by an increased airway surface tension.
Our data show that mild exercise-induced dehydration results in sustained, clinically significant reductions in FVC [>200 ml (32)] in the majority of athletes with mild asthma/EIB.Due to controversy over a potential impairment of airway secretions in individuals with asthma (23,29,30), our findings may not be applicable to all athletes.Nonetheless, considering the widespread prevalence of asthma/EIB in elite sport (13), the functional relevance of these findings deserves further attention.That end-expiratory lung volume decreases with exercise and that dehydration may affect peripheral airway stability at low lung volumes, it is tempting to speculate that exercise-induced dehydration may increase the risk of cyclic opening and closure of peripheral airways during exercise.In vitro, the re-opening of closed airways can cause epithelial injury (6).As repeated epithelial injury is regarded as a key mechanism in the pathogenesis of EIB in athletes (3), these findings could be highly relevant in the context of EIB.
In conclusion, whole-body dehydration does not exacerbate airway responsiveness in recreational athletes with mild asthma/EIB.However, lung volumes (incl.FVC, RV, FRC and RV/TLC) could be compromised in a state of mild dehydration.The functional and clinical relevance of these novel findings are yet to be established.

Figure 1 .
Figure 1.Schematic of protocol to assess changes in airway responsiveness and pulmonary function in a dehydration condition (2 h of exercise in the heat with fluid restriction), a control condition (2 h of exercise in ambient conditions with voluntary fluid consumption), and a time-matched rest condition (2 h of rest with voluntary fluid consumption).EVH, eucapnic voluntary hyperpnea; DLCO, diffusing capacity of the lung for carbon monoxide; V ̇E, ventilation; HR, heart rate.

Figure 2 .
Figure 2. Change in forced expiratory volume in 1 sec (FEV 1 ) after: i) exercise in a dehydrated state (dehydration), ii) exercise in a euhydrated state (control), and iii) a timematched rest condition (rest).Data are for ten recreational athletes with mild asthma and/or exercise-induced bronchoconstriction.Data are median and inter-quartile range.

Figure 3 .
Figure 3. Change in forced vital capacity (FVC) after: i) exercise in a dehydrated state (dehydration), ii) exercise in a euhydrated state (control), and iii) a time-matched rest condition (rest).Data are for ten recreational athletes with mild asthma and/or exerciseinduced bronchoconstriction.Data are mean ± 95% confidence interval.* P≤0.05, different vs. control and rest; ** P≤0.01 different vs. control and rest.Reduction in FVC >200 ml (---) is considered clinically meaningful (32).

Figure 4 .
Figure 4. Change in functional residual capacity (FRC) and residual volume (RV) after: i) exercise in a dehydrated state (dehydration), ii) exercise in a euhydrated state (control), and iii) a time-matched resting condition (rest).Data are for ten recreational athletes with mild asthma and/or exercise-induced bronchoconstriction.Data are mean ± 95% confidence interval.

Figure 5 .
Figure 5. Relationship between change in body mass and change in residual volume (RV) after 2 h of exercise with fluid restriction.

Table 1 .
Dynamic lung indices at baseline and after: i) exercise in a dehydrated state (dehydration), ii) exercise in a euhydrated state (control), and iii) a time-matched rest period (rest).P<0.05, different versus control at corresponding time point; R P<0.05, different versus rest at corresponding time point.

Table 2 .
Static lung volumes and capacities at baseline and after: i) exercise in a dehydrated state (dehydration), ii) exercise in a euhydrated state (control), and iii) a timematched rest period (rest).Data are mean ± SD for 10 participants.TLC, total lung capacity; FRC, functional residual capacity; RV, residual volume; ERV, expiratory reserve volume; * P<0.05, different versus baseline; C P<0.05, different versus control at corresponding time point; R P<0.05, different versus rest at corresponding time point.

Table 3 .
Indices of diffusing capacity at baseline and after: i) exercise in a dehydrated state (dehydration), ii) exercise in a euhydrated state (control) and iii) a time-matched rest period (rest).Data are mean ± SD for 9 participants.DLCO, diffusing capacity of the lung for carbon monoxide; KCO transfer coefficient, VA alveolar volume.